ORIGINAL RESEARCH published: 20 July 2021 doi: 10.3389/fcimb.2021.690731

The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria bassiana

Elen R. Muniz 1,2,Ca´ rita S. Ribeiro-Silva 2, Walqu´ıria Arruda 3, Nemat O. Keyhani 4 and E´ verton K. K. Fernandes 1,2*

1 Escola de Veterina´ ria e Zootecnia, Universidade Federal de Goia´ s, Goiaˆ nia, Brazil, 2 Instituto de Patologia Tropical e Sau´ de Pu´ blica, Universidade Federal de Goia´ s, Goiaˆ nia, Brazil, 3 Instituto de Cieˆ ncias Biolo´ gicas, Universidade Federal de Goia´ s, Goiaˆ nia, Brazil, 4 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, United States

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress–response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress–response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against

Edited by: Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout Guilhem Janbon, (DBbmsn2) and complemented mutant (DBbmsn2/Bbmsn2) strains. Reproductive Institut Pasteur, France parameters of R. microplus engorged females treated topically or by an intra-hemocoel Reviewed by: injection of conidial suspensions were assessed. Treated cuticles of engorged females Almudena Ortiz-Urquiza, Swansea University, were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was United Kingdom assessed. Topically treated engorged females showed high mean larval hatching (>84%) Hokyoung Son, D D Seoul National University, in control and Bbmsn2 treatments, whereas treatment with the wild-type or Bbmsn2/ South Korea Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. *Correspondence: Percent control of R. microplus topically treated with DBbmsn2 was lower than in the ´ Everton K. K. Fernandes groups treated with wild-type (56.1%) or DBbmsn2/Bbmsn2 strains. However, no [email protected] differences on reproductive parameters were detected when R. microplus were treated Specialty section: by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. This article was submitted to microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died Fungal Pathogenesis, a section of the journal before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed Frontiers in Cellular and similar conidial germination and formation of pseudo-appressoria on tick cuticle. Infection Microbiology Histological sections of ticks treated with the wild-type or DBbmsn2/Bbmsn2 strains Received: 04 April 2021 Accepted: 18 June 2021 showed fungal penetration through the cuticle, and into the tick interior. Hyphae Published: 20 July 2021 of DBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h Citation: after treatment. Protease activity was lower on tick cuticles treated with DBbmsn2 than Muniz ER, Ribeiro-Silva CS, Arruda W, those treated with the wild-type or DBbmsn2/Bbmsn2 strains. These data show that loss Keyhani NO and Fernandes E´ KK (2021) The Msn2 Transcription Factor of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but Regulates Acaricidal Virulence in the did not interfere with conidial germination, appressoria formation or sporulation on tick Fungal Pathogen Beauveria bassiana. Front. Cell. Infect. Microbiol. 11:690731. cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate doi: 10.3389/fcimb.2021.690731 that the BbMsn2 transcription factor acts mainly during the fungal penetration process

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and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.

Keywords: tick, biological control, entomopathogenic fungi, cuticle, virulence, Beauveria bassiana, Rhipicephalus microplus, Msn2

INTRODUCTION Urquiza and Keyhani, 2015). In entomopathogenic fungi, the transcription factor Msn2 regulates the conidiogenesis of Beauveria bassiana (Hypocreales: Cordycipitaceae) is one of the B. bassiana and Metarhizium robertsii; a knockout strain of each most widely studied entomopathogenic fungi for applied tick species (DBbmsn2 and DMrmsn2, respectively) reduced 43 and 39% control (Kirkland et al., 2004a; Kirkland et al., 2004b; Fernandes the conidial production in comparison to their respective wild-type et al., 2012). The potential of this fungus to control Rhipicephalus strains (Liu et al., 2013). Also, DBbmsn2 and DMrmsn2 strains had a microplus (Acari: Ixodidae) has been shown in laboratory assays, reduced cell tolerance to chemical and environmental stresses. with variable virulence among B. bassiana isolates (Posadas and Decreased virulence of DBbmsn2 and DMrmsn2 against Lecuona, 2009; Campos et al., 2010; Fernandes et al., 2011; Sun Spodoptera litura (Lepidoptera: Noctuidae) second-instar larvae et al., 2013). According to Fernandes et al. (2011), the mean and Tenebrio molitor (Coleoptera: Tenebrionidae) third-instar lethal concentration of B. bassiana to kill 50% (LC50)of larvae was recorded; the LT50 values were 28 and 25% longer R. microplus engorged female can vary from 107 to 109 than the control strains of B. bassiana (5.5 days) and M. robertsii − conidia ml 1. (4.8 days), respectively. In Galleria mellonella (Lepidoptera: fi B. bassiana conidia infect ticks through their cuticle or Pyralidae) larvae, the LT50 signi cantly increased when treated natural openings (Bernardo et al., 2018). The process of fungal with the knockout strain (LT50 =3.4±0.08days)incomparison infection on ticks is thought to be similar to that known for to the wild-type strain (2.71 ± 0.03 days) (Luo et al., 2015). These insects (Arruda et al., 2005), and includes conidial adhesion on data confirmed that Msn2 is critical for entomopathogenic fungal the host cuticle, production of germ tube, differentiation into infection of insects; however, any similar role towards Acari has not appressorium (something not seen for all B. bassiana isolates), been investigated. Furthermore, to date, no histological penetration through the host cuticle by enzymatic action (e.g., investigations related to Msn2 on infection or effects on lipases, proteases and chitinases) and mechanical pressure, and reproduction have been performed on insects or ticks. Overall, growth within the host integument and hemocoel (colonization little is known about the molecular basis of virulence in B. bassiana stage) (Ortiz-Urquiza and Keyhani, 2016). The host dies by towards ticks. Here, we examined the consequences of loss of Msn2 tissue destruction and by action of toxins from fungi (Schrank on B. bassiana infection towards ticks combining genetic and Vainstein, 2010). However, ticks are known to potentially characterization with enzymatic and histological approaches. Our display significantly higher natural resistances to insect data indicate BbMsn2 is critical for penetration but not subsequent pathogenic fungi, and acaricidal specificfactorsmaybe growth and proliferation once the tick cuticle has been breached. produced by these fungi (Kirkland et al., 2004a, Kirkland et al., Furthermore, important effects were seen with respect to lowered 2004b, Kirkland et al., 2005). female fertility, indicating potential added benefits in the application As mentioned, the virulence of B. bassiana is influenced by of B. bassiana for tick control. the production of proteases, particularly the subtilisin-like protease called Pr1 (Joshi et al., 1995), chitinases and lipases (Fang et al., 2005) and, specifically against ticks, the metabolite oxalic acid has been shown to be important (Kirkland et al., MATERIAL AND METHODS 2005). In the last fifteen years, many studies have contributed to understanding the network of genes which are related to the Beauveria bassiana Strains, Conidial virulence of B. bassiana (Jin et al., 2010; Ortiz-Urquiza and Suspensions, and Viability Keyhani, 2013; Wang et al., 2013; Valero-Jiménez et al., 2016). The strains of B. bassiana knockout DBbmsn2 and complemented Fang et al. (2005) demonstrated that overproduction of the (DBbmsn2/Bbmsn2) were constructed and initially characterized Bbchit1 chitinase enhanced the virulence of B. bassiana against by Luo et al. (2015). DBbmsn2 and the complemented strain were fi aphids, as indicated by the signi cantly lower LC50 and mean obtained from B. bassiana Bb0062. Beauveria bassiana strains lethal time to kill 50% (LT50) of target insects of the mutant strain were cultivated on potato dextrose agar (PDA, Difco Laboratories, − compared to the wild-type strain. Another study using hybrid Sparks, MD, USA) supplemented with 1 g L 1 yeast extract ™ chitinase gene (Bbchit1–BmChBD) showed a 23% reduction of (Bacto Yeast Extract, Sparks, MD, USA) (PDAY) in Petri the LT50 to kill aphids (Myzus persicae) when they were treated plates (90 × 15 mm) and incubated in the dark for 15 days at with the mutant strain of B. bassiana in comparison to its wild- 26 ± 1°C and relative humidity (RH) ≥90%. Temperature and type (Fan et al., 2007). relative humidity in the incubator were monitored with a data ® Transcriptional regulation of effector genes in eukaryotic cells logger HOBO H8 (Onset Computer Corporation, Bourne, MA, is one of the fundamental mechanisms involved in cellular USA). Fresh conidia from each strain were harvested using a ® responses to stress and/or virulence signaling pathways (Ortiz- spatula, suspended in 0.01% (v/v) Tween 80 (Labsynth Prod.

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Lab. Ltda, Diadema, SP, Brazil) and filtered through cheesecloth to The following reproductive parameters were investigated: remove mycelia. Conidial suspensions were quantified in estimated reproduction (ER) and percent control (PC) hemocytometer at 400× magnification in a Leica DM750 light (Drummond et al., 1971). The effectiveness of treatment was microscope (Leica Microsystems, Wetzlar, Germany), and the measured by the effect on the ER of the engorged females; in this − concentration was adjusted to 2.0 × 108 conidia ml 1 or as equation, 20,000 is the estimate of the number of larvae that indicated. To assess conidial viability, 20 µl of each conidial normally hatch from 1 g of eggs of R. microplus; therefore, the suspension were inoculated onto 8 ml of PDAY medium percent control of ER estimates the treatment efficacy to decrease supplemented with 0.002% (w/v) Benomyl (50% active the tick population in an infested environment. Bioassays were ® ingredient; Benlate , DuPont, São Paulo, SP, Brazil) (Braga repeated three times on different days, and with new batches of et al., 2001) and 0.05% (w/v) chloramphenicol (Sigma-Aldrich, conidia. The ER and PC were calculated by the Equations (1) and Steinheim, Germany) in Petri plates (35 × 9 ×10 mm). The plates (2), respectively. were incubated at 26 ± 1°C for 48 h. After incubation, a drop of Equation (1): lactophenol and cotton blue solution and coverslip were applied weight of egg mass (g) over the inoculum; a minimum of 300 conidia per plate was ER = evaluated, and the percent relative germination was determined initial weight of engorged female (g) (Braga et al., 2001). Conidia were considered germinated when the  percentage of larval hatch  20, 000 germ tube was longer than the maximum conidial diameter. Conidia were used only if viability was assessed to be >98% in Equation (2): all experiments. mean ER of the control group − mean ER of the treated group PC =  100 Fungal Bioassays Using Rhipicephalus mean ER of control group microplus and Measurement of Tick Reproductive Parameters Scanning Electron and Light Microscopy ’ Rhipicephalus microplus engorged females were collected from of Tick s Cuticle artificially infested cattle at Universidade Federal de Goiás (UFG, Initial steps of the B. bassiana infection process were examined Goiânia, Brazil). In the laboratory, ticks were washed in tap by scanning electron microscopy (SEM) and light microscopy. water, immersed in 1% (v/v) hypochlorite for 1 min, rinsed in Six R. microplus engorged females were individually treated with D D sterile distilled water for 1 min and dried with sterile paper BbWT, Bbmsn2/Bbmsn2 or Bbmsn2 by topical application of 8 −1 towels. The females were homogeneously distributed by weight 2.5 µl conidial suspension at 2.0 × 10 conidia ml . The treated ≥ (160-315 mg) into four treatment groups: control, BbWT, females were incubated at 26 ± 1°C, with RH 90% and 12 h fi DBbmsn2/Bbmsn2 and DBbmsn2; each group had 10 individuals. photophase, for 48 or 120 h. Then, approximately 50 µl of xative Two infection protocols were evaluated: (i) topical and (ii) [2% (v/v) glutaraldehyde (Impex, Labimpex Ind. Com. de Prods. intra-hemocoel injection. For topical assays, ticks were Lab. Ltda., Diadema, SP, Brazil), 2% (v/v) paraformaldehyde ® ı́ individually immersed in 1 ml of Tween 80 0.01% (control) (Vetec Qu mica Fina Ltda, Duque de Caxias, RJ, Brazil), 3% − or in the conidial suspensions at 2.0 × 108 conidia ml 1 for 3 min. (w/v) sucrose (Sigma-Aldrich, Steinheim, Germany) in 0.1 M In addition, ticks were individually inoculated by intra-hemocoel sodium cacodylate buffer (Sigma-Aldrich, Steinheim, Germany), ® injection with 5 µl of Tween 80 0.01% (control), or 5 µl of pH 7.2] was injected into each female using an insulin syringe − conidial suspension at 1.6 × 105 conidia ml 1 (800 conidia/tick), according to Barreto et al. (2016). Each female was placed in a − fi 1.6 × 106 conidia ml 1 (8,000 conidia/tick), 1.6 × 107 conidia 15-ml centrifuge tube containing 2 ml of xative and maintained − − ml 1 (80,000 conidia/tick) or 2.0 × 108 conidia ml 1 (106 conidia/ for 10 days at 4°C in a refrigerator. tick); injections were performed in the foramen located between Ticks incubated for 48 h were examined by SEM (n = 3). The the capitulum and the dorsal scutum of engorged females using a dorsal cuticle of females was dissected and removed, and then stereomicroscope and a 0.3 mm insulin syringe (Angelo et al., washed three times (15 min each time) in sodium cacodylate 2010). After treatment, ticks were individually placed in each buffer (0.1 M, pH 7.2). Cuticles were dehydrated in a graded well of 24-well cell culture plates (Corning Brasil Indústria series of ethanol solutions (30, 50, 70, 80 and 90%), held for e Comércio Ltda., Suzano, SP, Brazil), incubated at 26 ± 1°C, 15 min in each solution and passed twice in 100% ethanol for RH ≥90% and 12 h photophase. The egg mass from each female 15 min. Subsequently, the cuticles were individually placed in was collected at the end of oviposition, weighed, transferred to an micro-centrifuge tubes containing 300 µl of hexamethyldisilazane fi individual glass tube (16 × 125 mm), closed with a cotton plug, (Electron Microscopy Sciences, Hat eld, PA, USA) and and incubated at 26 ± 1°C and RH ≥90% for assessment of larval maintained immersed for 5 min (Barreto et al., 2016). After hatching. The eggs were observed daily and the larval hatching drying, the samples proceeded to metallization. Accordingly, the for each tube was visually estimated through microscopic samples were placed on a stub and coated with gold in a sputter- examination, and values were assigned in percentages ranging applicator (Denton Vacuum Desk V). The cuticles were analyzed from 0 to 100% by intervals of 5% in relation to the total mass of and electro micrographs were obtained with a scanning electron eggs (Drummond et al., 1971; Barreto et al., 2016; Bernardo microscope (Jeol JSM 6610) at an accelerating voltage of 20 kV. et al., 2018). The images were analyzed qualitatively (conidial adhesion, size of

Frontiers in Cellular and Infection Microbiology | www.frontiersin.org3 July 2021 | Volume 11 | Article 690731 Muniz et al. BbMsn2 Regulates Virulence to Ticks germinative tubes and presence of appressoria) and quantitatively Statistical Analyses (number of germinated conidia). All data sets were previously checked for normality and Ticks incubated for 120 h were prepared for histological homoscedasticity with Shapiro–Wilk and Bartlett tests, analyses of their cuticle. After 10 d in fixative, treated ticks respectively. Normally distributed data (engorged female initial (n = 3) were longitudinally cut, dehydrated as described fi ® weight, egg mass weight, and conidial germination) were tted to with SEM samples and embedded in resin (Historesin , Leica a parametric model and then were submitted to ANOVA followed Biosystems, Wetzlar, Germany) according to the manufacturer’s by an SNK test for multiple comparisons. Non-normally distributed instructions. Sections of 4 µm were made in Microtome (Leica data (percentage of larval hatch) were fitted to a non-parametric Biosystems, Wetzlar, Germany), stained with Periodic Acid- model and then analyzed by a Kruskal–Wallis test, followed by an Schiff (PAS) and Green light (Arruda et al., 2005) and assessed FDR test. Protease activity of mutant strains was compared with the by using a light microscope (Nikon E200). Images (n = 5) were BbWT strain by applying the Dunnett’s test. Analyses were captured with a high-definition microscope camera Leica ICC50 performed in the statistical environment R (R Team C, 2018). P- HD, with resolution of 1,280 × 720 p (HD ready). The values less than 0.05 were considered as significant. experiments were conducted three times on different days with three replicates in each treatment group. RESULTS Protease Assay Cuticles of R. microplus engorged females were dissected (n = 10), Measurement of Reproductive Parameters washed in distilled water and immersed in 10 ml conidial and Virulence Assays suspension of the strain BbWT, DBbmsn2/Bbmsn2 or DBbmsn2, Reproductive parameters of R. microplus engorged females treated − at 2.0 × 108 conidia ml 1 for 3 min. Cuticles were incubated at 26°C topically with different strains (BbWT, DBbmsn2,andDBbmsn2/ and RH ≥90% for 120 h. After incubation, the pool of cuticles was Bbmsn2)ofB. bassiana were examined 15 d post-infection after − macerated in 1 ml distilled water and centrifuged at 25,000 RCF for immersion in 2.0 × 108 conidia ml 1 as detailed in the Material and 5 min at 4°C. Protease activity was measured by azocasein Methods section (Table 1). Because of the difficulty in determining hydrolysis as described by Segers et al. (1994) and Phillips et al. the exact timing of tick death, lethal mortality times could not be (1984) with the following modifications: commercial azocasein accurately determined; however, reproductive parameters that (Sigma Chemical Co., St. Louis, MO, USA) was dissolved at 1% would critically inform on successful biological control efforts (w/v) in 0.1 M Tris–HCl buffer, pH 8.5. Briefly, 1,000 µl of each were measured. The weight of egg mass from females treated with supernatant sample were incubated with 500 µl de azocasein 1% at any of the B. bassiana conidial suspensions tested (i.e., either wild- 28°C for 60 min. This reaction was stopped by adding 1 ml of 10% type, mutant, or complemented mutant) was similar and lower TCA and maintaining it for 15 min at 4°C, and followed by (~50%) than that of control untreated ticks (F3,118 = 18.33; centrifugation at 25,000 RCF for 10 min to remove the P<0.001). High mean larval hatching (~90%) was observed in precipitated protein. Six hundred milliliters of the supernatant the control group, which was slightly reduced after treatment with were neutralized by adding 700 µl of 1M NaOH, and absorbance the DBbmsn2 strain (down to ~84%, P = 0.006). However, engorged at 450 nm was recorded with Enzyme-Linked Immunosorbent females treated with BbWT or DBbmsn2/Bbmsn2 had significantly Assay (ELISA). One unit of enzyme activity (U) was calculated by decreased larval hatching (10–20%, c2 = 17.38; df =3;P =0.006), the Equation (3): indicating lower egg viability using the wild-type and Equation (3): complemented B. bassiana strains. Overall, the percent control of ticks was higher in the groups treated with BbWT (56.1%) or control absorbance − sample absorbance 1 DBbmsn2/Bbmsn2 (58.7%) as compared to untreated engorged UA = Â 0:001 60 females or ticks treated with the DBbmsn2 mutant strain (39.7%).

TABLE 1 | Biological parameters of Rhipicephalus microplus engorged females treated by immersion in Tween 80® 0.01% (control) or in conidial suspension (2.0 × 108 conidia ml−1)ofBeauveria bassiana strains (BbWT, DBbmsn2/Bbmsn2 or DBbmsn2) incubated at 26 ± 1°C and RH ≥ 90%.

Treatment by immersion Engorged female weight (mg) Egg mass weight (mg) Larval hatch (%) ER† Percent control (%)‡ (n = 30)

Control 232.1 ± 16a 126.3 ± 15a 89.2 ± 3.2a 968,730.1 – BbWT 231.2 ± 16a 66.4 ± 13b 78.5 ± 3.0bc 447,094.8 56.1 DBbmsn2/Bbmsn2 233.4 ± 14a 68.2 ± 15b 70.9 ± 4.9c 406,020.6 58.7 DBbmsn2 232.5 ± 16a 77.6 ± 16b 84.0 ± 2.2ab 554,807.5 39.7

(†) ER: estimated reproduction [ER = weight of egg mass (g)/initial weight of engorged female (g) × percentage of larval hatch × 20,000] (Drummond et al., 1971). (‡) Percent control [ER of the control group − mean ER of the treated group/mean ER of the control group × 100] (Drummond et al., 1971). Means are followed by standard errors of 10 replicates per bioassay, in three independent trials. Means followed by the same letter in the same column did not differ significantly (P > 0.05) according to the Student–Newman–Keuls (engorged female weight and egg mass weight) or Kruskal–Wallis test (larval hatch).

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Despite being unable to determine exact time of death, all ticks appeared to have germinated, and appressoria-like tip structures showed eventual mycosis and sporulation of the fungus on the could be seen. tick cadavers. Conidia from BbWT and DBbmsn2/Bbmsn2 were capable of For intra-hemocoel injection assays (that would bypass the germinating and penetrating through the cuticle (Figures 4A, C, need to cuticle penetrations), a concentration range of fungal respectively). Hyphae of BbWT (Figures 4A, B) and DBbmsn2/ conidia (800, 80,000, 80,000, and 106 conidia/tick) was tested Bbmsn2 were also observed inside the tick body, with fungal (Table 2). Engorged females of R. microplus treated by intra- development in adjacent tissues (Figures 4C, D). However, the hemocoel injection of 800 conidia/tick showed decreased (50– hyphae of the DBbmsn2 strain appeared to continue to grow on 60%) mean egg mass weights for all strains tested (F3,55 = 24.05; the surface/exocuticle of the tick with fewer instances of P<0.001) as compared to control uninfected ticks, with only penetrative hypha seen at 120 h incubation as compared to the slight differences seen between the wild-type, DBbmsn2, and wild-type and complemented strains (Figure 4E). DBbmsn2/Bbmsn2 strains (Table 2). In addition, a decreased mean oviposition period from 7 to 2 d was seen after injection of Protease Activity 800 conidia/tick of any of the B. bassiana strains tested (F3,55 = The proteolytic activity of the B. bassiana strains on R. microplus 27.66; P<0.001) in comparison to control untreated ticks. Ticks cuticle was measured using azoalbumin as detailed in the Material treated by intra-hemocoel injection at concentrations ≥8,000 and Methods section. Protease activity was significantly lower in − conidia/tick died before laying their eggs (i.e., within 48 h after the DBbmsn2 strain (1.91 ± 0.29 U ml 1) than in the BbWT (9.88 ± − treatment), while in control group the oviposition period was 4.08 U ml 1; P = 0.0113) and DBbmsn2/Bbmsn2 strains (8.16 ± − 7.9 ± 1 d (Table 2), and thus had essentially no oviposition 1.59 U ml 1; P = 0.0150) (Figure 5). period. No differences in sporulation were observed on the cadaver of ticks between the wild-type, DBbmsn2,and DBbmsn2/Bbmsn2 strains at 5 d after infection (Figure 1). DISCUSSION

Infection of B. bassiana Strains on Our data indicate that the Msn2 transcription factor significantly Tick Cuticle contributes to the ability of B. bassiana to infect R. microplus via the The absence of the Msn2 transcription factor did not interfere “natural” cuticle-penetration requiring route. Engorged females with conidial germination or adhesion to the tick cuticle. treated topically with the DBbmsn2 strain showed a significantly Electron micrographs showed similar (F2,6 = 0.0638; P = decreased percent control of ticks in comparison to the wild-type 0.9388) conidial germination between the B. bassiana strains and complemented strains. However, the mortality of ticks reached on the tick cuticle at 48 h incubation: BbWT = 84.33 ± 13.2%, 100% when engorged females were injected, thus by-passing the DBbmsn2/Bbmsn2 = 82.33 ± 11.20% and DBbmsn2 = 76.33 ± requirement for cuticle penetration, with DBbmsn2 (≥8,000 conidia/ 22.67% (Figure 2). Most conidia from BbWT (Figure 3A), tick) conidial suspension by intra-hemocoel injection. Even at the DBbmsn2/Bbmsn2 (Figures 3B, C) and DBbmsn2 (Figure 3D) lowest dose injected (800 conidia/tick), reduced reproductive

TABLE 2 | Biological parameters of Rhipicephalus microplus engorged females treated by an intra-hemocoel injection with 5 µl of Tween 80® 0.01% (control), or 5 µl of conidial suspension of Beauveria bassiana strains (BbWT, DBbmsn2/Bbmsn2 and DBbmsn2) at 1.6 × 105 conidia ml−1 (800 conidia/tick), 1.6 × 106 conidia ml−1 (8,000 conidia/tick), 1.6 × 107 conidia ml−1 (80,000 conidia/tick) or 2.0 × 108 conidia ml−1 (106 conidia/tick), and incubated at 26 ± 1°C and RH ≥ 90%.

Treatment by inoculation Dosage (conidia/tick) Engorged female weight (mg) Egg mass weight (mg) Oviposition period (days)*

Control 0 249.1 ± 0.6a 78.1 ± 19 (n = 30) 7.9 ± 1 BbWT 106 249.0 ± 1.1a 0(n=0) – DBbmsn2/Bbmsn2 106 250.4 ± 1.6a 0(n=0) – DBbmsn2 106 251.6 ± 0.9a 0(n=0) – Control 0 236.1 ± 6.0a 76.4 ± 3.9 (n = 30) 7.9 ± 0.4 BbWT 8×104 239.3 ± 6.6a 0(n=0) – DBbmsn2/Bbmsn2 8×104 237.5 ± 5.7a 0(n=0) – DBbmsn2 8×104 232.3 ± 6.4a 0(n=0) – Control 0 235.6 ± 6.8a 77.8 ± 5 (n = 30) 6.7 ± 0.1 BbWT 8×103 239.2 ± 7.0a 0(n=0) – DBbmsn2/Bbmsn2 8×103 236.4 ± 7.8a 0(n=0) – DBbmsn2 8×103 237.1 ± 6.6a 0(n=0) – Control 0 237.9 ± 8.2a 76.8 ± 5a (n = 30) 7.2 ± 0.5a BbWT 800 237.6 ± 9.1a 32.7 ± 6b (n = 7) 2.3 ± 0.1b DBbmsn2/Bbmsn2 800 239.6 ± 9.0a 11.0 ± 1b (n = 11) 2.0 ± 0.0b DBbmsn2 800 238.9 ± 9.0a 14.6 ± 5b (n = 12) 2.1 ± 0.05b

(*) Mean number of days engorged females laid their eggs. Means are followed by standard errors of 30 replicates per bioassay. Means followed by the same letter in the same column, and in the same dosage tested, did not differ significantly (P > 0.05) according to the Student–Newman–Keuls test.

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FIGURE 1 | Fungal sporulation on Rhipicephalus microplus engorged females’ cadavers at day 5 post treatment by an intra-hemocoel injection with 5-ml conidial suspension at 2.0 × 108 conidia ml–1. parameters of engorged females was seen for all of the B. bassiana penetration of B. bassiana through the tick cuticle, with retention strains tested. In a previous study, S. litura and T. molitor larvae of the ability of the fungus to evade immune systems once inside D treated with Bbmsn2 conidia had, respectively, LT50 values 28 and the host. 25% higher (decreased virulence) than the complemented strain The first step to successful infection of entomopathogenic fungi (Liu et al., 2013). In addition, G. mellonella larvae treated topically is the adhesion of fungal propagules (conidia or blastospores) on D with Bbmsn2 conidia also had an increased LT50 detected; the host cuticle. Accordingly, high fungal virulence is directly however, no difference in LT50 values among the mutant and related to increased adhesion to the host cuticle (Butt and Goettel, wild-type strains was observed when G. mellonella larvae were 2000). Adhesion is considered to follow a two-step process treated by injecting conidial suspension into the hemocoel (Luo involving initial attachment followed by consolidation of et al., 2015). These results and our data lead us to conclude that the adhesion, influenced by the surface characteristics of the fungal transcription factor Msn2 acts predominantly during the cells and the target substrata (Holder et al., 2007). The B. bassiana conidial surface is composed of a layer of hydrophobic rod-like proteins (hydrophobins, hyd1 and hyd2), which mediate, in part, attachment to the host cuticle that is also hydrophobic (Holder and Keyhani, 2005; Ortiz-Urquiza and Keyhani, 2016). The deletion of hyd1 gene in B. bassiana decreased the conidia hydrophobicity and virulence, although it did not interfere in its adhesion capacity. The deletion of the hyd2 gene caused changes in the structure of the conidia cell wall, and consequently diminished its adhesion capacity and hydrophobicity but without altering its virulence (Zhang et al., 2011). Liu et al. (2013) have shown that the hydrophobin genes (hyd1 and hyd2) were repressed by 66–68% in a DBbmsn2 mutant strain, but it did not affect the mad1 and mad2 expressions, additional factors important for facilitating conidial adhesion to arthropods and plants, respectively (Broetto et al., 2010). Microscopic analyses were used in order to probe effects of BbMsn2 on adhesion to tick D FIGURE 2 | Germination (%) of conidia of Beauveria bassiana strains: BbWT, surfaces, and these data showed that conidia of Bbmsn2 were DBbmsn2/Bbmsn2 and DBbmsn2 on the cuticle of Rhipicephalus microplus equivalent to the wild-type (BbWT) and complemented strains engorged females incubated at 26 ± 1°C and RH ≥90% for 48 h. Bars with (DBbmsn2/Bbmsn2) in size and ability to adhere to the tick cuticle. the same letter do not differ significantly (P ≥ 0.05) among treatments. These data indicate that the impaired virulence seen in topical Standard errors are based on three independent trials. assays is not likely due to impaired adhesion to the host surface.

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FIGURE 3 | Electron micrographs of Rhipicephalus microplus engorged females treated topically with conidia of Beauveria bassiana strains: BbWT, DBbmsn2/ Bbmsn2 or DBbmsn2, and incubated for 48 h at 26 ± 1°C and RH ≥ 90%. Germinating conidia of BbWT on the tick cuticle (A) Germinating conidia of DBbmsn2/ Bbmsn2 with developed appressorium on the tick cuticle (B) Germinating conidia of DBbmsn2/Bbmsn2 on the tick cuticle (C) Germinated conidia of DBbmsn2 [some with long germ tubes (hyphae)] on the tick cuticle (D). CO, conidia; AP, appressorium; H, hyphae.

The tick cuticle is divided in several layers; from the outside to protease activity can vary significantly according to the inside: epicuticle, exocuticle, endocuticle and epidermis composition of host cuticle (Cito et al., 2016), which may (Hackman, 1982). The epicuticle layer is composed of lipids, induce or inhibit fungal development (Ment et al., 2012). long chain alkenes, esters and fatty acids (Ali et al., 2009). In Additionally, Santi et al. (2019) identified 50 proteins involved particular, R. microplus females, during the engorgement in the infection process, which were produced by B. bassiana process, have altered epicuticle composition that includes an cultured in media supplemented with cuticles derived from R. almost 20% increase in surface lipids; with the most commonly microplus, but not produced in media supplemented with found including alcohol and fatty acids combined by glucose only. Our results showed a decreased penetration of esterification (Hackman, 1982). To penetrate the host cuticle, DBbmsn2 hyphae through the cuticle in comparison to the wild- entomopathogenic fungi use mechanical pressure and secrete type and complemented strains at 120 h post-infection. This proteases, chitinases, and lipases, which degrade their main inability to trespass the tick cuticle may be due to the reduced constituents (proteins, chitin, and lipids) to allow the hyphae capacity to degrade chitin, proteins, and/or lipids that constitute to penetrate through the exoskeleton. B. bassiana secretes lipases the cuticle composition of R. microplus (Hackman, 1982). In which potentiate the degradation of the arthropod wax layer addition, impairments related to the fungal response to the (Sánchez-Pérez et al., 2014). However, Feng (1998) indicated that osmotic, oxidative and/or nutrient stress that occurs on the lipase activity of several B. bassiana s.l. isolates had little cuticle, and are mediated by Msn2, may also contribute to correlation with their virulence. Luo et al. (2015) demonstrated the decreased virulence (Luo et al., 2015). that proteinase and lipase activities were similar in DBbmsn2 The production of oxalic acid is also an important virulence colonies grown on skim milk agar plates and in the wild-type. factor in B. bassiana against ticks. Direct treatment of ticks with However, in the present study, our data show that the protease oxalate at pH 4.0 resulted in almost 80% mortality in adults of activity of B. bassiana DBbmsn2 on R. microplus cuticle decreased the tick Amblyomma americanum within 14 days after treatment in the Msn2 mutant. It is known that the production of (total) (Kirkland et al., 2005). Oxalate production reduces extracellular

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FIGURE 4 | Sagittal sections of Rhipicephalus microplus engorged females treated with conidia of Beauveria bassiana strains: BbWT, DBbmsn2/Bbmsn2 or DBbmsn2, and incubated for 120 h at 26 ± 1°C and RH ≥90%. Germinating conidia of BbWT penetrates through the tick cuticle (A) Germinating conidia of BbWT attaches to the tick cuticle and fungal hyphae infecting the tick interior (B) Germinating conidia of DBbmsn2/Bbmsn2 penetrates through all the layers of the tick cuticle (C) Hyphae of DBbmsn2/Bbmsn2 infects the interior tick tissues (D) Incomplete penetration of DBbmsn2 hyphae through the tick cuticle (E). Co, germinated conidia; H, hyphae inside tick tissue; asterisk (*) cuticle. pH and, consequently, acts to facilitate degradation of however its contribution to infection may be limited (Fan et al., components of the host cuticle, e.g., chitin, elastin and collagen 2017). A critical difference with respect to Msn2 function (Bidochka and Khachatourians, 1991). M. anisopliae mutants between insects and ticks may be that in insects Msn2 appears unable to acidify the culture medium (i.e., that produced less to contribute to both topical and intra-hemocoel infection (Luo metabolic acids) also show a decreased protease activity (St Leger et al., 2015), whereas our data indicate that for ticks, Msn2 is et al., 1999). In B. bassiana, Luo et al. (2015) demonstrated that essentially only required for full virulence via the topical route of the DBbmsn2 strain had little to no radial growth at pH 4.1 or 4.7 infection. While topical infection is the “natural” route of (although conidiation still occurred), and its growth was reduced infection, these data imply important downstream immune in comparison to the wild-type strain. In addition, oosporein system differences in dealing with invading (fungi) microbes production is impaired in the Msn2 mutant (Luo et al., 2015), between ticks and insects.

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differentiation, and even fungal colonization on the host surface may be poor indicators of successful tick control (mortality), which requires host penetration (Ment et al., 2012).

CONCLUSIONS

Our results indicate that the absence of Msn2 transcription factor reduced the virulence of B. bassiana s.l. against R. microplus demonstrated by the delayed fungal penetration and decreased protease production on the tick cuticle. Results on tick reproduction revealed potential effects beyond direct virulence that can impact biological control efforts.

DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be FIGURE 5 | Protease activity on the cuticle of Rhipicephalus microplus treated by an immersion in a conidial suspension of Beauveria bassiana strains: BbWT, made available by the authors, without undue reservation. DBbmsn2/Bbmsn2 or DBbmsn2, and incubated for 120 h at 26 ± 1°C and RH ≥90%. Bars with different letters, i.e. “a” and “b”, indicate significant difference (P < 0.05). Standard errors are based on three independent trials. ETHICS STATEMENT

The animal study was reviewed and approved by Ethics Commission on Animal Use of Universidade Federal de Goiás Effects on reproduction can be critical to the biological control (CEUA, protocol #057/16). The access to Brazilian genetic potential of insect pathogenic fungi. Reduced fecundity and lack of heritage was approved by the Genetic Heritage Management resistance development have been reported in using Cordyceps Council (CGen) of Brazil (protocol #A420934). fumosorosea (formerly, Isaria fumosorosea)againstBemisia tabaci (whitefly) (Gao et al., 2017), as well as in B. bassiana (Bernardo et al., 2018)orM. anisopliae (Bittencourt et al., 1994; Muniz et al., AUTHOR CONTRIBUTIONS 2020) infecting R. microplus engorged females. After penetrating ́ the cuticle of R. microplus, M. anisopliae reaches the hemocoel EM, NK and EF designed the experiments and wrote the manuscript. and can be found colonizing the hemolymph (Bittencourt et al., EM and CR-S performed the bioassays, histology of ticks and protease 1995) and internal organs, including the ovary tissues (Paulo activity assays. EM and WA performed scanning electron microscopy et al., 2018) thus contributing to the reduced fecundity of and histology of ticks. NK inspired co-authors to investigate this subject engorged females seen after infection. B. bassiana infection and produced the mutant strains of B. bassiana. All authors contributed towards Argas persicus (Acari: Argasidae) also directly affects to the article and approved the submitted version. the female reproductive system and causes damages to the ovaries, inhibiting vitellogenesis (Marzouk et al., 2020). Our data show that B. bassiana infection reduced fecundity of R. FUNDING microplus engorged females by reducing ovipositing and larval This study was supported by the Coordenação de Aperfeiçoamento hatching, with loss of Msn2 impairing these effects. For de Pessoal de Nıveĺ Superior (CAPES) of Brazil for providing PhD successful control, even if entomopathogenic fungi may not scholarship for EM and CR-S. This research was supported by (quickly) kill engorged females, any reduction in fecundity can grants from the Conselho Nacional de Desenvolvimento Cientıfíco be crucial for controlling R. microplus infestations, because fully eTecnológico (CNPq) of Brazil (431928/2016-9 to ÉF). CNPq also engorged females naturally drop off the host and lay thousands of provided the grant 306319/2018-7 for ÉF. This research was also eggs on the ground before dying and completing their life cycle. supported in part by US-NSF grant IOS-1557704 to NK. The In recent years, studies on the identification of genes and their publication costs were covered by grant from the Fundação de functions in B. bassiana have revealed a wide range of Amparo à Pesquisa do Estado de Goiás (FAPEG; CC 11233). mechanisms involved in the infection process (Butt et al., 2016); however, most of these studies have focused on insect larvae that are naturally more susceptible to infection by these ACKNOWLEDGMENTS fungi (Ortiz-Urquiza and Keyhani, 2016), and the extent to which these results can be extrapolated to other targets within We thank Dr. Filippe Elias de Freitas Soares (Visiting professor Arthropoda remains to be determined. Our data indicate some at UFG, and current professor at Universidade Federal de shared contributions as well as potential differences. In addition, Lavras), and MSc. Juliana Marques Ferreira (PhD student at our results indicate that conidial germination, appressorial UFG) for teaching EM the protocol used for the protease assays.

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